The present study investigates the potential of SARS-CoV-2 inactivation by a copper sulfide (CuS) incorporated three-layer mask design. The mask consisted of the outer, middle, and inner layers to give comfort, strength, shape, and safety. The outer layer contained a total of 4.4 % CuS (w/w) (2.2% CuS coated & 2.2 % CuS impregnated) nylon fibers and the middle entrapment area contain a total of 17.6% CuS (w/w) impregnated nylon. No CuS was present in the inner layer. The antiviral efficacy assessment revealed, CuS incorporated mask is highly effective in inactivating SARS-CoV-2 within 30 min exposure. After, 1h and 2 h exposure, near-complete elimination of virus were observed by cytopathy, fluorescence, and viral copy number. The antiviral activity of the mask material was derived by incorporated solid-state CuS. Noticeably, the antiviral activity of CuS against SARS-CoV-2 was in the form of solid-state CuS, but not as Cu 2+ ionic form derived by dissolved CuSO 4 . The kinetics of droplet entrapment revealed, that the three-layered mask almost completely block virus-containing droplet pass-through for short exposure periods of 1 to 2 min, and 80% efficacy for longer exposure times of 5 to 10 min. We also demonstrated the incorporated CuS is evenly distributed all over the fibers assuring the uniformity of potential antiviral activity and proves, CuS particles are not easily shed out of the fabric fibers. The inactivation efficacy demonstrated against SARS-CoV-2 proves that the CuS incorporated three-layer mask will be a lifesaver during the present intense global pandemic.

Chitosan nanoparticles (CNPs) represent an efficient vaccination tool to deliver immunogenic antigens to the antigen-presenting cells (APCs), which subsequently stimulate protective immune responses against infectious diseases. Herein, we prepared CNPs encapsulating mRNA molecules followed by surface coating with conserved H9N2 HA2 and M2e influenza proteins. We demonstrated that CNPs efficiently delivered mRNA molecules into APCs and had effectively penetrated the mucosal barrier to reach to the immune initiation sites. To investigate the potential of CNPs delivering influenza antigens to stimulate protective immunity, we intranasally vaccinated chickens with empty CNPs, CNPs delivering HA2 and M2e in both mRNA and protein formats (CNPs + RNA + Pr) or CNPs delivering antigens in protein format only (CNPs + Pr). Our results demonstrated that chickens vaccinated with CNPs + RNA + Pr elicited significantly (p < 0.05) higher systemic IgG, mucosal IgA antibody responses and cellular immune responses compared to the CNPs + Pr vaccinated group. Consequently, upon challenge with either H7N9 or H9N2 avian influenza viruses (AIVs), efficient protection, in the context of viral load and lung pathology, was observed in chickens vaccinated with CNPs + RNA + Pr than CNPs + Pr vaccinated group. In conclusion, we show that HA2 and M2e antigens elicited a broad spectrum of protection against AIVs and incorporation of mRNAs in vaccine formulation is an effective strategy to induce superior immune responses.

Introduction The emergence of SARS-CoV-2 variants has raised concerns on future vaccine efficacy as most vaccines target only the spike protein. Hence, vaccines targeting multiple SARS-CoV-2 proteins will offer broader protection and improve our preparedness to combat the pandemic. Objectives The study aimed to develop a novel vaccine strategy by combining a eukaryotic vector expressing multiple SARS-CoV-2 genes and Salmonella -mediated in vivo DNA delivery. Methods The eukaryotic vector was designed to function as a DNA-launched RNA replicon in a self-replicating and self-amplifying mRNA mechanism. By exploiting the self-cleaving peptide, P2A, we fused four SARS-CoV-2 targets, including receptor-binding domain (RBD), heptad repeat domain (HR), membrane protein (M) and epitopes of nsp13, in a single open reading frame. Western blot and immunofluorescence assays were used to determine protein expression. In mice, the vaccine's safety and immunogenicity were investigated. Results Western blot analysis revealed co-expression all four proteins from the vaccine construct, confirming the efficiency of Salmonella -mediated gene delivery and protein expression. The vaccine candidate was safe and elicited robust antigen-specific antibody titers in mice, and a recall response from splenocytes revealed induction of strong cell-mediated immunity. Flow cytometry demonstrated an increase in sub-populations of CD4 + and CD8 + T cells with the highest CD4 + and CD8 + T cells recorded for HR and RBD, respectively. Overall, humoral and cellular immune response data suggested the induction of both Th1 and Th2 immunity with polarization towards an antiviral Th1 response. SARS-CoV-2 neutralization assays exhibited potent neutralizing antibody titers in mouse immune sera. Conclusions The Salmonella bactofection ensured optimum in vivo gene delivery, and through a P2A-enabled efficient multicistronic expression, the vaccine candidate elicited potent anti-SARS-CoV-2 immune responses. These findings provide important insight into development of an effective multivalent vaccine to combat SARS-CoV-2 and its variants.

The ongoing SARS-CoV-2 evolution has resulted in many variants, contributing to the striking drop in vaccine efficacy and necessitated the development of next-generation vaccines to tackle antigenic diversity. Herein we developed a multivalent Semliki Forest virus replicon-based mRNA vaccine targeting the receptor binding domain (RBD), heptad repeat domain (HR), membrane protein (M) and epitopes of nsp13 of SARS-CoV-2. The bacteria-mediated gene delivery offers the rapid production of large quantities of vaccine at a highly economical scale and notably allows the needle-free mass vaccination. A favourable Th1 dominated potent antibody and cellular immune responses were detected in the immunized mice. Further, immunization induced strong cross-protective neutralizing antibodies (NAbs) against the B.1.617.2 delta variant (Clade G). We recorded a difference in induction of IgA response by the immunization route with the oral route eliciting a strong mucosal sIgA response, which possibly has contributed to the enhanced protection conferred by the oral immunization. Hamsters immunized orally were completely protected against the viral replication in the lungs and the nasal cavity. Importantly, the vaccine protected the hamsters against SARS-CoV-2-induced pneumonia. The study provides proof-of-principle findings for the development of a feasible and efficacious oral mRNA vaccine against SARS-CoV-2 and its variants.

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